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Alkaline phosphatase activity --- secreted

1. Transfect cells with plasmids encoding heat stable secreted alkaline phosphatase (AP) gene as reporter.

2. 24-48 hours after transfection, harvest 200 ml of medium.

3. Heat the medium to 65 C for 30 minutes to inactivate any endogenous phosphatases.

4. Spin down cell debris.

5. Transfer 50 ml to 96-well plate wells.

6. Add 150 l of substrate (5 M p-nitrophenyl phosphate (PNPP) in 50 mM Tris-HCl, pH 8.2) to each well.

Adding 25 l 0.05% Zwittergent (in PBS) may reduce background.

7. If quantitative measurment is required, use an AP standard to generate a standard curve.

8. Incubate at room temperature or 37 C for 30 minutes or more.

9. Read the plate at 405 nm.